Joseph Riss

The contribution of VHL substrate binding and HIF1-α to the phenotype of VHL loss in renal cell carcinoma

Clear-cell renal carcinoma is associated with inactivation of the von Hippel-Lindau (VHL) tumor suppressor gene. VHL is the substrate recognition subunit of an E3 ligase, known to target the alpha subunits of the HIF heterodimeric transcription factor for ubiquitin-mediated degradation under normoxic conditions. We demonstrate that competitive inhibition of the VHL substrate recognition site with a peptide derived from the oxygen degradation domain of HIF1alpha recapitulates the tumorigenic phenotype of VHL-deficient tumor cells. These studies prove that VHL substrate recognition is essential to the tumor suppressor function of VHL. We further demonstrate that normoxic stabilization of HIF1alpha alone, while capable of mimicking some aspects of VHL loss, is not sufficient to reproduce tumorigenesis, indicating that it is not the critical oncogenic substrate of VHL.

Predicting survival in patients with metastatic kidney cancer by gene-expression profiling in the primary tumor

To identify potential molecular determinants of tumor biology and possible clinical outcomes, global gene-expression patterns were analyzed in the primary tumors of patients with metastatic renal cell cancer by using cDNA microarrays. We used grossly dissected tumor masses that included tumor, blood vessels, connective tissue, and infiltrating immune cells to obtain a gene-expression “profile” from each primary tumor. Two patterns of gene expression were found within this uniformly staged patient population, which correlated with a significant difference in overall survival between the two patient groups. Subsets of genes most significantly associated with survival were defined, and vascular cell adhesion molecule-1 (VCAM-1) was the gene most predictive for survival. Therefore, despite the complex biological nature of metastatic cancer, basic clinical behavior as defined by survival may be determined by the gene-expression patterns expressed within the compilation of primary gross tumor cells. We conclude that survival in patients with metastatic renal cell cancer can be correlated with the expression of various genes based solely on the expression profile in the primary kidney tumor.

Regulation of HIF prolyl hydroxylases by hypoxia‐inducible factors

Hypoxia and induction of hypoxia‐inducible factors (HIF‐1α and HIF‐2α) is a hallmark of many tumors. Under normal oxygen tension HIF‐alpha subunits are rapidly degraded through prolyl hydroxylase dependent interaction with the von Hippel‐Lindau (VHL) tumor suppressor protein, a component of E3 ubuiquitin ligase complex. Using microarray analysis of VHL mutated and re‐introduced cells, we found that one of the prolyl hydroxylases (PHD3) is coordinately expressed with known HIF target genes, while the other two family members (PHD1 and 2) did not respond to VHL. We further tested the regulation of these genes by HIF‐1 and HIF‐2 and found that siRNA targeted degradation of HIF‐1α and HIF‐2α results in decreased hypoxia‐induced PHD3 expression. Ectopic overexpression of HIF‐2α in two different cell lines provided a much better induction of PHD3 gene than HIF‐1α. In contrast, we demonstrate that PHD2 is not affected by overexpression or downregulation of HIF‐2α. However, induction of PHD2 by hypoxia has HIF‐1‐independent and ‐dependent components. Short‐term hypoxia (4 h) results in induction of PHD2 independent of HIF‐1, while PHD2 accumulation by prolonged hypoxia (16 h) was decreased by siRNA‐mediated degradation of HIF‐1α subunit. These data further advance our understanding of the differential role of HIF factors and putative feedback loop in HIF regulation. Published 2004 Wiley‐Liss, Inc.

Cancers as Wounds that Do Not Heal: Differences and Similarities between Renal Regeneration/Repair and Renal Cell Carcinoma

Cancers have been described as wounds that do not heal, suggesting that the two share common features. By comparing microarray data from a model of renal regeneration and repair (RRR) with reported gene expression in renal cell carcinoma (RCC), we asked whether those two processes do, in fact, share molecular features and regulatory mechanisms. The majority (77%) of the genes expressed in RRR and RCC were concordantly regulated, whereas only 23% were discordant (i.e., changed in opposite directions). The orchestrated processes of regeneration, involving cell proliferation and immune response, were reflected in the concordant genes. The discordant gene signature revealed processes (e.g., morphogenesis and glycolysis) and pathways (e.g., hypoxia-inducible factor and insulin-like growth factor-I) that reflect the intrinsic pathologic nature of RCC. This is the first study that compares gene expression patterns in RCC and RRR. It does so, in particular, with relation to the hypothesis that RCC resembles the wound healing processes seen in RRR. However, careful attention to the genes that are regulated in the discordant direction provides new insights into the critical differences between renal carcinogenesis and wound healing. The observations reported here provide a conceptual framework for further efforts to understand the biology and to develop more effective diagnostic biomarkers and therapeutic strategies for renal tumors and renal ischemia.

Mistaken Identifiers: Gene name errors can be introduced inadvertently when using Excel in bioinformatics

BackgroundWhen processing microarray data sets, we recently noticed that some gene names were being changed inadvertently to non-gene names.ResultsA little detective work traced the problem to default date format conversions and floating-point format conversions in the very useful Excel program package. The date conversions affect at least 30 gene names; the floating-point conversions affect at least 2,000 if Riken identifiers are included. These conversions are irreversible; the original gene names cannot be recovered.ConclusionsUsers of Excel for analyses involving gene names should be aware of this problem, which can cause genes, including medically important ones, to be lost from view and which has contaminated even carefully curated public databases. We provide work-arounds and scripts for circumventing the problem.

Deletions of chromosomes 3p and 14q molecularly subclassify clear cell renal cell carcinoma

The short arm of chromosome 3 (3p) harbors the von Hippel‐Lindau (VHL) tumor suppressor gene, and the long arm of chromosome 14 (14q) harbors the hypoxia‐inducible factor 1α (HIF‐1α) gene. The objective of this study was to evaluate the significance of 3p loss (loss VHL gene) and 14q loss (loss HIF‐1α gene) in clear cell renal cell carcinoma (ccRCC).

Gain of Chromosome 8q Is Associated With Metastases and Poor Survival of Patients With Clear Cell Renal Cell Carcinoma

The aim of this study was to evaluate the prevalence of chromosome 8q gain in clear cell renal cell carcinoma (CCRCC) and to correlate the findings with tumor phenotype and disease‐specific survival (DSS).

Dendritic cell-based immunotherapy in prevention and treatment of renal cell carcinoma: efficacy, safety, and activity of Ad-GM·CAIX in immunocompetent mouse models.

The dendritic cell vaccine DC-Ad-GM·CAIX is an active, specific immunotherapy with the potential of providing a safe and effective therapy against renal cell carcinoma (RCC). Using immunocompetent Balb/c mouse models we tested the efficacy and mechanism of the vaccine to prevent and treat the growth of a syngeneic RCC (RENCA) engineered to overexpress the human TAA carbonic anhydrase IX (NPR-IX). In a prevention model, NPR-IX tumor development was specifically and significantly delayed by 13 days in DC-Ad-GM·CAIX-treated mice (P<0.001), tumor volumes were 79% smaller (day 24, P<0.007), and body weight was maintained at study termination compared with the controls. Six of these mice remained tumor-free for >1 year. In a treatment model, NPR-IX tumors remained smaller in DC-Ad-GM·CAIX-treated mice for 8 days (P<0.002), achieving a 60% growth inhibition at termination. No vaccine-related organ toxicity was observed in either model. The critical mechanistic parameter separating responsive from nonresponsive tumors was hCAIX protein expression, demonstrated by aggressive growth of tumors that did not express hCAIX protein and in sham-treated mice (DC-Ad-Null). No murine serum anti-hCAIX antibodies were detected. Moreover, altered mechanisms of immunoediting as a means for immune evasion were suggested by differential gene expression (Ccl1, Hmgb1, Fgl2, Cd209a, and Klra2) and therapy evasion miRNAs (miR-1186, miR-98, miR-5097, miR-1942, and miR-708) in tumors that evaded DC-Ad-GM·CAIX immunotherapy. This is the first study in immunocompetent mice that provides a proof of concept for the specificity, efficacy, safety, and activity of the DC-Ad-GM·CAIX immunotherapy, forming the basis for a first-in-human phase I trial in RCC patients.

Salvage-targeted kidney cancer therapy in patients progressing on high-dose interleukin-2 immunotherapy: the UCLA experience.

PurposeTo analyze the outcomes of patients with metastatic renal cell carcinoma treated with salvage-targeted therapy after progressing on high-dose interleukin (IL)-2 immunotherapy in a tertiary referral center. Materials and MethodsA retrospective nonrandomized cohort consisting of 286 patients with metastatic renal cell carcinoma treated from 2003 to 2010 was analyzed from the University of California, Los Angeles (UCLA) Kidney Cancer database. All patients underwent cytoreductive nephrectomy, and 21 patients received salvage-targeted therapy after progression on high-dose IL-2, whereas 111 patients received targeted therapy alone. The remaining 154 patients had other treatment combinations or experimental targeted therapy agents only. Since 2003, selection of patients for high-dose IL-2 was increasingly based on clinical, pathologic, and molecular criteria (UCLA CPM criteria). Disease-specific survival was calculated from diagnosis of metastatic renal cell carcinoma. ResultsPatients selected according to UCLA CPM criteria and treated with salvage-targeted therapy after progressing on high-dose IL-2 experienced a significantly greater disease-specific survival (median not reached) than those treated with targeted therapy alone (30 months; P = 0.004). Since 2006, all high-dose IL-2 patients met the UCLA CPM criteria and were able to receive salvage-targeted therapy upon progression. Disease-specific survival calculated from initiation of targeted therapy was comparable for patients treated with salvage-targeted therapy after progression on high-dose IL-2 (34 months) versus first-line targeted therapy (26 months; P = 0.175). DiscussionPatients selected for high-dose IL-2 based on UCLA CPM criteria and treated with salvage-targeted therapy upon progression have achieved outstanding disease-specific survival. Our data suggest a new algorithm for carefully selected patients with metastatic renal cell carcinoma based on UCLA CPM criteria to receive first-line high-dose IL-2 while reserving their option for salvage-targeted therapy with uncompromised efficacy upon progression.

Abstract 2819: Efficacy and safety of the Ad-GM·CAIX dendritic cell-based vaccine in treating in vivo metastatic renal cell carcinoma compared to sunitinib monotherapy and simultaneous vaccine-sunitinib combination therapy

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Metastatic clear cell renal cell carcinoma (ccRCC) disease is responsible for significant morbidity and represents the main cause of death in patients with advanced ccRCC. We have developed a novel dendritic cell based vaccine targeting human carbonic anhydrase IX (hCAIX; DC-Ad-GM·CAIX) and compared it with the ccRCC standard-of-care drug, sunitinib as a monotherapy and in simultaneous vaccine-sunitinib combination therapy. Immunocompetent mice (Balb/c) were orthotopically-transplanted with syngeneic RCC-hCAIXpositive (NPR-IX) tumor cells, immunized, and/or treated with sunitinib at low-dose (5mg/kg/d), high-dose (40mg/kg/d) or untreated. At termination, primary tumor size (weight), lung metastatic burden, hCAIX and immune-markers expression levels were compared. Mono-immunotherapy with DC-Ad-GM·CAIX vaccine suppressed metastatic tumor growth: the total number of metastatic heterotypic foci (i.e., hCAIX positive and negative foci) following vaccination decreased 2.5 fold compared with untreated mice (P 10 fold). Vaccination alone resulted in reduced primary tumor burden of RCC-hCAIXpositive cells to <25% of the tumor cell population, with the remaining cells lacking hCAIX expression (hCAIXnegative); there was no significant overall primary tumor reduction compared to untreated mice. In contrast, sunitinib, whether given as high-dose monotherapy or in combination with the vaccine, inhibited the primary orthotopic tumors, achieving 35% (P=0.0001) and 51% (P=1.7e-7) tumor reduction, respectively. However, sunitinib monotherapy was less effective in reducing the metastatic tumor burden compared with the vaccine. In fact, simultaneous administration of vaccine and sunitinib increased the metastatic tumor burden in heterotypic tumors composed also of hCAIXnegative cells. In conclusion, this preclinical study demonstrates that (i) the DC-Ad-GM·CAIX vaccine effectively controls both primary and metastatic hCAIXpositive tumors and forms the basis for a phase I trial in metastatic ccRCC patients, which has been initiated at UCLA (http://clinicaltrials.gov number [NCT01826877][1]), (ii) Antigen editing with loss of hCAIX is an important immune escape mechanism, (iii) This in vivo study raises caution regarding the use of DC-Ad-GM·CAIX vaccine in simultaneous combination therapies with sunitinib. Co-corresponding Authors: ASB and JR Citation Format: Edward N. Rampersaud, Jonathan W. Said, Adrian Bot, Frederic D. Birkhauser, Nils Kroeger, Gang Zeng, Fairooz F. Kabbinavar, Antoni Ribas, Allan J. Pantuck, Arie S. Belldegrun, Joseph Riss. Efficacy and safety of the Ad-GM·CAIX dendritic cell-based vaccine in treating in vivo metastatic renal cell carcinoma compared to sunitinib monotherapy and simultaneous vaccine-sunitinib combination therapy. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2819. doi:10.1158/1538-7445.AM2014-2819 [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01826877&atom=%2Fcanres%2F74%2F19_Supplement%2F2819.atom